Connective tissue growth factor(CTGF)

ABSTRACT

A novel chemotactic and mitogenic protein, Connective Tissue Growth Factor (CTGF), a polynucleotide that encodes CTGF and antibodies that bind to CTGF are provided. Diagnostic and therapeutic methods using CTGF are also described.

This is a continuation of application Ser. No. 07,752,427, filed on Aug.30, 1991, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to the field of growth factors andspecifically to Connective Tissue Growth Factor (CTGF) and the geneencoding this factor.

2. Related Art

Growth factors are a class of secreted polypeptides that stimulatetarget cells to proliferate, differentiate and organize in developingtissues. The action of growth factors is dependent on their binding tospecific receptors which stimulates a signalling event within the cell.Examples of some well-studied growth factors include platelet-derivedgrowth factor (PDGF), insulin-like growth factor (IGF-I), transforminggrowth factor beta (TGF-β), transforming growth factor alpha (TGF-α),epidermal growth factor (EGF), and fibroblast growth factor (FGF).

PDGF is a cationic, heat-stable protein found in the alpha-granules ofcirculating platelets and is known to be a mitogen and a chemotacticagent for connective tissue cells such as fibroblasts and smooth musclecells. Because of the activities of this molecule, PDGF is believed tobe a major factor involved in the normal healing of wounds andpathologically contributing to such diseases as atherosclerosis andfibrotic diseases. PDGF is a dimeric molecule consisting of an A chainand a B chain. The chains form heterodimers or homodimers and allcombinations isolated to date are biologically active.

Studies on the role of various growth factors in tissue regeneration andrepair have led to the discovery of PDGF-like proteins. These proteinsshare both immunological and biological activities with PDGF and can beblocked with antibodies specific to PDGF.

These new growth factors may play a significant role in the normaldevelopment, growth, and repair of human tissue. Therapeutic agentsderived from these molecules may be useful in augmenting normal orimpaired growth processes involving connective tissues in certainclinical states, e.g., wound healing. When these growth factors areinvolved pathologically in diseases, therapeutic developments from theseproteins may be used to control or ameliorate uncontrolled tissuegrowth.

The isolation of these factors and the genes encoding them is importantin the development of diagnostics and therapeutics for variousconnective tissue-related disorders. The present invention provides suchan invention.

SUMMARY OF THE INVENTION

Various cell types produce and secrete PDGF and PDGF-related molecules.In an attempt to identify the type of PDGF dimers present in the growthmedia of cultured endothelial cells, a new growth factor was discovered.This previously unknown factor, termed Connective Tissue Growth Factor(CTGF), is related immunologically and biologically to PDGF, however itis the product of a distinct gene.

In a first aspect, the present invention relates to a polypeptide growthfactor for connective tissue cells. The polypeptide is mitogenic and isalso a chemotactic agent for cells.

In a second aspect, the present invention provides a polynucleotideencoding a connective tissue growth factor characterized as encoding aprotein (1) existing as a monomer of approximately 36-38 kD molecularweight, and (2) capable of binding to a PDGF receptor.

In a further aspect, the invention provides a method for acceleratingwound healing in a subject by applying to the wound an effective amountof a composition which contains purified CTGF.

In yet another aspect, the invention provides a method of diagnosingpathological states in a subject suspected of having pathologycharacterized by a cell proliferative disorder which comprises, (1)obtaining a sample suspected of containing CTGF from the subject, (2)determining the level of CTGF in the sample, and (3) comparing the levelof CTGF in the sample to the level of CTGF in normal tissues.

A method of ameliorating diseases characterized by a cell proliferativedisorder, by treating the site of the disease with an effective amountof a CTGF reactive agent is also provided.

DETAILED DESCRIPTION OF THE INVENTION

The present invention discloses a novel protein growth factor calledConnective Tissue Growth Factor (CTGF). This protein may play asignificant role in the normal development, growth and repair of humantissue. The discovery of the CTGF protein and cloning of the cDNAencoding this molecule is significant in that it is a previously unknowngrowth factor having mitogenic and chemotactic activities for connectivetissue cells. The biological activity of CTGF is similar to that ofPDGF, however, CTGF is the product of a gene unrelated to the A or Bchain genes of PDGF. Since CTGF is produced by endothelial andfibroblastic cells, both of which are present at the site of a wound, itis probable that CTGF functions as a growth factor in wound healing.Pathologically, CTGF may be involved in diseases in which there is anovergrowth of connective tissue cells, such as cancer, fibrotic diseasesand atherosclerosis. The CTGF polypeptide could be useful as atherapeutic in cases in which there is impaired healing of skin woundsor there is a need to augment the normal healing mechanisms.Additionally, antibodies to CTGF polypeptide or fragments could bevaluable as diagnostic tools to aid in the detection of diseases inwhich CTGF is a pathological factor. Therapeutically, antibodies orfragments of the antibody molecule could also be used to neutralize thebiological activity of CTGF in diseases where CTGF is inducing theovergrowth of tissue.

The primary biological activity of CTGF polypeptide is its mitogenicity,or ability to stimulate target cells to proliferate. The ultimate resultof this mitogenic activity in vivo, is the growth of targeted tissue.CTGF also possesses chemotactic activity, which is the chemicallyinduced movement of cells as a result of interaction with particularmolecules. Preferably, the CTGF of this invention is mitogenic andchemotactic for connective tissue cells, however, other cell types maybe responsive to CTGF polypeptide as well.

The CTGF polypeptide of the invention is characterized by existing as amonomer of approximately 36-38 kD molecular weight. CTGF is secreted bycells and is active upon interaction with a PDGF receptor of cells. CTGFis antigenically related to PDGF although there is little if any peptidesequence homology. Anti-PDGF antibody has high affinity to thenon-reduced forms of the PDGF isomers and the CTGF molecule and ten-foldless affinity to the reduced forms of these peptides, which lackbiological activity. This suggests that there are regions of sharedtertiary structure between the PDGF isomers and the CTGF molecule,resulting in common antigenic epitopes.

The term "substantially pure" as used herein refers to CTGF which issubstantially free of other proteins, lipids, carbohydrates or othermaterials with which it is naturally associated. The substantially purepolypeptide will yield a single major band on a non-reducingpolyacrylamide gel. The purity of the CTGF polypeptide can also bedetermined by amino-terminal amino acid sequence analysis. CTGFpolypeptide includes functional fragments of the polypeptide, so long asthe mitogenic and chemotactic activities of CTGF are retained. Smallerpeptides containing the biological activity of CTGF are included in theinvention. Additionally, more effective CTGF molecules produced, forexample, through site directed mutagenesis of the CTGF cDNA areincluded.

The invention provides polynucleotides encoding the CTGF protein. Thesepolynucleotides include DNA, cDNA and RNA sequences which encodeconnective tissue growth factor. It is understood that allpolynucleotides encoding all or a portion of CTGF are also includedherein, so long as they encode a polypeptide with the mitogenic andchemotactic activity of CTGF. Such polynucleotides include bothnaturally occurring and intentionally manipulated polynucleotides. Forexample, CTGF polynucleotide may be subjected to site-directedmutagenesis. The polynucleotides of the invention include sequences thatare degenerate as a result of the genetic code. There are only 20natural amino acids, most of which are specified by more than one codon.Therefore as long as the amino acid sequence of CTGF is functionallyunchanged, all degenerate nucleotide sequences are included in theinvention.

The sequence of the cDNA for CTGF contains an open reading frame of 1047nucleotides with an initiation site at position 130 and a TGAtermination site at position 1177 and encodes a peptide of 349 aminoacids. There is only a 40% sequence homology between the CTGF cDNA andthe cDNA for both the A and B chains of PDGF.

The CTGF open reading frame encodes a polypeptide which contains 39cysteine residues, indicating a protein with multiple intramoleculardisulfide bonds. The amino terminus of the peptide contains ahydrophobic signal sequence indicative of a secreted protein and thereare two N-linked glycosylation sites at asparagine residues 28 and 225in the amino acid sequence. There is a 45% overall sequence homologybetween the CTGF polypeptide and the polypeptide encoded by the CEF-10mRNA transcript (Simmons, et al., Proc. Natl. Acad. Sci. USA 86:1178,1989); the homology reaches 52% when a putative alternative splicingregion is deleted.

DNA sequences of the invention can be obtained by several methods. Forexample, the DNA can be isolated using hybridization procedures whichare well known in the art. These include, but are not limited to: 1)hybridization of probes to genomic or cDNA libraries to detect sharednucleotide sequences and 2) antibody screening of expression librariesto detect shared structural features.

Screening procedures which rely on nucleic acid hybridization make itpossible to isolate any gene sequence from any organism, provided theappropriate probe is available. For example, oligonucleotide probes,which correspond to a part of the sequence encoding the protein inquestion, can be synthesized chemically. This requires that short,oligopeptide, stretches of amino acid sequence must be known. The DNAsequence encoding the protein can be deduced from the genetic code,however, the degeneracy of the code must be taken into account. It ispossible to perform a mixed addition reaction when the sequence isdegenerate. This includes a heterogeneous mixture of denatureddouble-stranded DNA. For such screening, hybridization is preferablyperformed on either single-stranded DNA or denatured double-strandedDNA. Hybridization is particularly useful in the detection of cDNAclones derived from sources where an extremely low amount of mRNAsequences relating to the polypeptide of interest are present. In otherwords, by using stringent hybridization conditions directed to avoidnon-specific binding, it is possible, for example, to allow theautoradiographic visualization of a specific cDNA clone by thehybridization of the target DNA to that single probe in the mixturewhich is its complete complement (Wallace, et al., Nucleic AcidResearch, 9:879, 1981 ).

A cDNA expression library, such as lambda gt11, can be screenedindirectly for CTGF peptides having at least one epitope, usingantibodies specific for CTGF or antibodies to PDGF which cross reactwith CTGF. Such antibodies can be either polyclonally or monoclonallyderived and used to detect expression product indicative of the presenceof CTGF cDNA.

DNA sequences encoding CTGF can be expressed in vitro by DNA transferinto a suitable host cell. "Host cells" are cells in which a vector canbe propagated and its DNA expressed. The term also includes any progenyof the subject host cell. It is understood that all progeny may not beidentical to the parental cell since there may be mutations that occurduring replication. However, such progeny are included when the term"host cell" is used.

DNA sequences encoding CTGF can be expressed in vivo in eitherprokaryotes or eukaryotes. Methods of expressing DNA sequences havingeukaryotic coding sequences in prokaryotes are well known in the art.Hosts include microbial, yeast and mammalian organisms.

Biologically functional viral and plasmid DNA vectors capable ofexpression and replication in a host are known in the art. Such vectorsare used to incorporate DNA sequences of the invention. In general,expression vectors containing promotor sequences which facilitate theefficient transcription of the inserted eukaryotic genetic sequence areused in connection with the host. The expression vector typicallycontains an origin of replication, a promoter, and a terminator, as wellas specific genes which are capable of providing phenotypic selection ofthe transformed cells.

In addition to expression vectors known in the art such as bacterial,yeast and mammalian expression systems, baculovirus vectors may also beused. One advantage to expression of foreign genes in this invertebratevirus expression vector is that it is capable of expression of highlevels of recombinant proteins, which are antigenically and functionallysimilar to their natural counterparts. Baculovirus vectors and theappropriate insect host cells used in conjunction with the vectors willbe known to those skilled in the art. The isolation and purification ofhost cell expressed polypeptides of the invention may be by anyconventional means such as, for example, preparative chromatographicseparations and immunological separations such as those involving theuse of monoclonal or polyclonal antibody.

Transformation of the host cell with the recombinant DNA may be carriedout by conventional techniques well known to those skilled in the art.Where the host is prokaryotic, such as E. coli, competent cells whichare capable of DNA uptake can be prepared from cells harvested afterexponential growth and subsequently treated by the CaCl₂ method usingprocedures well known in the art. Alternatively, MgCl₂ or RbCl could beused.

Where the host used is a eukaryote, various methods of DNA transfer canbe used. These include transfection of DNA by calciumphosphate-precipitates, conventional mechanical procedures such asmicroinjection, insertion of a plasmid encased in liposomes, or the useof virus vectors.

Eukaryotic host cells may also include yeast. For example, DNA can beexpressed in yeast by inserting the DNA into appropriate expressionvectors and introducing the product into the host cells. Various shuttlevectors for the expression of foreign genes in yeast have been reported(Heinemann, J. et al., Nature, 340:205, 1989; Rose, M. et al., Gene,60:237, 1987).

The invention provides antibodies which are specifically reactive withCTGF polypeptide or fragments thereof. Although this polypeptide iscross reactive with antibodies to PDGF, not all antibodies to CTGF willalso be reactive with PDGF. Antibody which consists essentially ofpooled monoclonal antibodies with different epitopic specificities, aswell as distinct monoclonal antibody preparations are provided.Monoclonal antibodies are made from antigen containing fragments of theprotein by methods well known in the art (Kohler, et al., Nature,256:495, 1975; Current Protocols in Molecular Biology, Ausubel, et al.,ed., 1989). Monoclonal antibodies specific for CTGF can be selected, forexample, by screening for hybridoma culture supernatants which reactwith CTGF, but do not react with PDGF.

The invention provides a method for accelerating wound healing in asubject, e.g., human, by applying to the wound an effective amount of acomposition which contains purified CTGF. PDGF and PDGF-relatedmolecules, such as CTGF, are involved in normal healing of skin wounds.The CTGF polypeptide of this invention is valuable as a therapeutic incases in which there is impaired healing of skin wounds or there is aneed to augment the normal healing mechanisms, e.g., burns. Oneimportant advantage to using CTGF protein to accelerate wound healing isattributable to the molecule's high percentage of cysteine residues.CTGF, or functional fragments thereof, is more stable and lesssusceptible to protease degradation than PDGF and other growth factorsknown to be involved in wound healing.

CTGF is produced by endothelial cells and fibroblastic cells, both ofwhich are present at the site of a skin wound. Therefore, agents whichstimulate the production of CTGF can be added to a composition which isused to accelerate wound healing. Preferably, the agent of thisinvention is transforming growth factor beta. The composition of theinvention aids in healing the wound, in part, by promoting the growth ofconnective tissue. The composition is prepared by combining, in apharmaceutically acceptable carrier substance, e.g., inert gels orliquids, the purified CTGF and TGF-β.

The term "cell proliferative disorder" refers to pathological statescharacterized by the continual multiplication of cells resulting in anovergrowth of a cell population within a tissue. The cell populationsare not necessarily transformed, tumorigenic or malignant cells, but caninclude normal cells as well. For example, CTGF may be involvedpathologically by inducing a proliferative lesion in the intimal layerof an arterial wall, resulting in atherosclerosis. Instead of trying toreduce risk factors for the disease, e.g., lowering blood pressure orreducing elevated cholesterol levels in a subject, CTGF inhibitors orantagonists of the invention would be useful in interfering with the invivo activity of CTGF associated with atherosclerosis. CTGF antagonistsare useful in treating other disorders associated with overgrowth ofconnective tissues, such as various fibrotic diseases, includingscleroderma, arthritis, alcoholic liver cirrhosis, keloid, hypertropicscar.

The present invention provides a method to detect the presence ofelevated levels of CTGF to be used diagnostically to determine thepresence of pathologies characterized by a cell proliferative disorder.For example, a sample suspected of containing CTGF is obtained from asubject, the level of CTGF determined and this level is compared withthe level of CTGF in normal tissue. The level of CTGF can be determinedby immunoassays using anti-CTGF antibodies, for example. Othervariations of such assays which are well known to those skilled in theart, such as radioimmunoassay (RIA), ELISA and immunofluorescence canalso be used to determine CTGF levels in a sample. Alternatively,nucleic acid probes can be used to detect and quantitate CTGF mRNA forthe same purpose.

The invention also discloses a method for ameliorating diseasescharacterized by a cell proliferative disorder by treating the site ofthe disease with an effective amount of a CTGF reactive agent. The term"ameliorate" denotes a lessening of the detrimental effect of thedisease-inducing response in the patient receiving therapy. Where thedisease is due to an overgrowth of cells, an antagonist of CTGFpolypeptide is effective in decreasing the amount of growth factor thatcan bind to a CTGF specific receptor on a cell. Such an antagonist maybe a CTGF specific antibody or functional fragments thereof (e.g., Fab,F(ab')₂). The treatment requires contacting the site of the disease withthe antagonist. Where the cell proliferative disorder is due to adiminished amount of growth of cells, a CTGF reactive agent which isstimulatory is contacted with the site of the disease. For example,TGF-β is one such reactive agent. Other agents will be known to thoseskilled in the art.

When a cell proliferative disorder is associated with the expression ofCTGF, a therapeutic approach which directly interferes with thetranslation of CTGF messages into protein is possible. For example,antisense nucleic acid or ribozymes could be used to bind to the CTGFmRNA or to cleave it. Antisense RNA or DNA molecules bind specificallywith a targeted gene's RNA message, interrupting the expression of thatgene's protein product. The antisense binds to the messenger RNA forminga double stranded molecule which cannot be translated by the cell.Antisense oligonucleotides of about 15-25 nucleotides are preferredsince they are easily synthesized and have an inhibitory effect justlike antisense RNA molecules. In addition, chemically reactive groups,such as iron-linked ethylenediaminetetraacetic acid (EDTA-Fe) can beattached to an antisense oligonucleotide, causing cleavage of the RNA atthe site of hybridization. These and other uses of antisense methods toinhibit the in vitro translation of genes are well known in the art(Marcus-Sakura, Anal., Blochem., 172:289, 1988).

The following examples are intended to illustrate but not limit theinvention. While they are typical of those that might be used, otherprocedures known to those skilled in the art may alternatively be used.

EXAMPLE 1 IDENTIFICATION AND PARTIAL PUBIFICATION OF MITOGEN FROM HUVECELLS PDGF-IMMUNORELATED Cells

Human umbilical vein endothelial (HUVE) cells were isolated from freshhuman umbilical cords by collagenase perfusion (Jaffe, et al., HumanPathol., 18:234, 1987) and maintained in medium 199 with 20% FCS, 0.68mM L-glutamine, 20 μg/ml Gentamicin, 90 μg/ml porcine heparin (Sigma,St. Louis, Mo.), and 50 μg/ml Endothelial Cell Growth Supplement(Sigma).

Cells used for media collection were third passage cells. Cells wereidentified as endothelial cells by their non-overlapping cobblestonemorphology and by positive staining for Factor-VIII related antigen. NRKcells were obtained from American Type Culture, NIH/3T3 cells were agift from S. Aaronson (NCI, Bethesda, Md.), and both cell lines weremaintained in DMEM, 10% FCS, 20 μg/ml Gentamicin. Fetal bovine aorticsmooth muscle cells were obtained from tissue explants as previouslydescribed (Grotendorst, et al., Proc. Natl. Acad. Sci. USA, 78:3669,1981) and maintained in DMEM, 10% FCS, 20 μg/ml Gentamicin, and used inassays at second or third passage.

Growth Factors and Antibodies

Human PDGF was purified to homogeneity from platelets as describedpreviously (Grotendorst, Cell, 36:279, 1984). Recombinant AA, BB, and ABchain dimeric PDGF molecules were obtained from Creative Biomolecules,(Hopkinton, Mass.). FGF was obtained from Sigma. Purified PDGF orsynthetic peptides containing the amino and carboxyl sequences of themature PDGF A and B chain molecules were used to raise antibodies ingoats. Goats were immunized with 20 μg of purified PDGF or 50 μg ofsynthetic peptide in Freunds complete adjuvant by multiple intradermalinjections. Immune sera were collected seven days after the fourthrechallenge (in Freunds incomplete adjuvant) and subsequentrechallenges. The anti-PDGF antibody did not show any cross-reactivityto TGF-β, EGF, or FGF in immunoblot analysis. The anti-peptideantibodies were sequence specific and did not cross-react with othersynthetic peptide sequences or with recombinant PDGF peptides which didnot contain the specific antigenic sequence. This was determined byWestern blot and dot blot analysis.

Antibody Affinity Column

Goat anti-human PDGF IgG (150 mg) was covalently bound to 25 mls ofAffi-Gel 10 support (BioRad) according to the manufacturers instructionswith a final concentration of 6 mg IgG/ml gel. The column was incubatedwith agitation at 4° C. for 18 hours with 1 liter of HUVE cell mediawhich had been conditioned for 48 hours. The gel was then poured into acolumn (5×1.5 cm), washed with four volumes of 0.1N acetic acid made pH7.5 with ammonium acetate, and the antibody-bound PDGF immunoreactiveproteins eluted with 1N acetic acid. Peak fractions were determined bybiological assays and immunoblotting and the fractions pooled.

Initial studies of the PDGF-related growth factors secreted by HUVEcells were done by removing the serum containing growth media fromconfluent cultures of cells and replacing it with serum-free media.Aliquots of this media were removed periodically and the proteinsimmunoblotted using an antibody specific for human platelet PDGF. Thisantibody does not cross-react with any other known growth factors and isable to detect less than 500 picograms of dimeric PDGF or 10 nanogramsof reduced, monomeric A or B chain peptide on immunoblots. HUVE cellswere grown to confluence in 6 well plates. The growth media was removed,cells washed with PBS and 1 ml of serum-free media was added to eachwell. The media was removed after conditioning for the period of timefrom 6-48 hours, dialyzed against 1N acetic acid and lyophilized. Thesamples were then run on 12% PAGE, electroblotted to nitrocellulose andvisualized with the anti-human PDGF antibody. Five nanograms of purifiedplatelet PDGF was run as reference.

The results indicated constitutive secretion of several species ofmolecules which are immunologically similar to platelet PDGF but are ofhigher relative molecular weight (36-39 kD) than the expected 30-32 kDMW of platelet PDGF or A chain or B chain homodimers. Chemotactic andmitogenic assays performed with this serum-free conditioned mediaindicated the total biological activity present was equivalent to 15ng/ml of platelet PDGF after a 48 hour conditioning period. Incubationof the media with 30 μg/ml of anti-human PDGF IgG neutralizedapproximately 20-30% of the mitogenic activity and similar amount of thechemotactic activity.

The presence in HUVE culture media of several species of PDGFimmunoreactive molecules was unexpected, particularly molecules ofhigher molecular weight than those of the A and B chain dimericmolecules anticipated to be produced and secreted by endothelial cells(Collins, et al., Nature, 328:621-624, 1987; Sitaras, et al., J. Cell.Physiol., 132:376-380, 1987). In order to obtain greater amounts of thePDGF-like proteins for further analysis, the HUVE cells had to be keptin media containing 20% fetal calf serum, since the cells begin to dieafter 24 hours in serum-free or low serum media. The PDGF immunoreactiveproteins were partially purified from the serum containing media by useof an antibody affinity column made with the anti-human PDGF IgG and anAffi-Gel 10 support (BioRad). Mitogenic assays were performed using NRKcells as target cells (PDGF BB =5 ng/ml, PDGF AA=10 ng/ml). HUVE mediawas 250 μl of HUVE cell serum-free conditioned media (48 hours) whichwas dialyzed against 1N acetic acid, lyophilized, and resuspended inDMEM before addition to test wells. Affinity purified fraction was 5μl/ml of combined, concentrated major pool from Affi-Gel 10 affinitycolumn. Anti-PDGF IgG or non-immune IgG (30 μg/ml) was added to thesamples and incubated 18 hours at 4° C. prior to testing in themitogenic assay. The mean of triplicate samples was determined and thestandard deviation was less than 5%. The experiments were repeated atleast three times with similar results.

When aliquots of the partially purified proteins were assayed forchemotactic and mitogenic activity, all biological activity could beneutralized by prior incubation of the proteins with the anti-human PDGFantibody. This indicated that the only biologically active moleculespresent in the partially purified media proteins were PDGF immunorelatedmolecules.

Aliquots of the partially purified proteins were immunoblotted using thesame anti-PDGF antibody and the data indicated the presence of thehigher MW molecules observed in the serum-free conditioned media. Themajor species secreted migrates on polyacrylamide gels at 36 kD andcomprises at least 50% of the total immunoreactive protein purified fromconditioned media. The immunoreactive species migrating at 37 and 39 kDconstitute most of the remaining immunoreactive protein. A similarpattern was seen with proteins labeled with ³⁵ S-cysteine and affinitypurified with the anti-PDGF IgG immunoaffinity column. Less than 15% ofthe total affinity purified proteins co-migrated with purified plateletPDGF or recombinant PDGF isoforms.

Prior incubation of the antibody with purified PDGF (300 ng PDGF/2 μgIgG) blocked antibody binding to all of the molecules, indicating sharedantigenic determinants with dimeric platelet PDGF. Interestingly, whenthe antibody was blocked with recombinant AA, BB, or AB dimers, antibodybinding to the HUVE secreted proteins was inhibited equally by all threedimeric forms, suggesting that the antibody recognizes common epitopespresent on all three PDGF dimers and the HUVE secreted molecules. Inorder to insure that none of the antibody binding molecules detected onWestern blots was derived from fetal calf serum or other additives inthe culture media, a new, unused antibody affinity column was made andmedia which was never conditioned by cells was processed exactly as theconditioned media. No PDGF immunoreactive molecules were detected in thefractions from this column by immunoblot and no biological activity wasdetected. When platelet PDGF or the recombinant dimers are reduced with200 mM dithiothreitol (DTT), monomeric A chain (17 kD) and B chain (14kD) peptides are observed on immunoblots. Treating the HUVE molecules ina 100 mM DTT sample buffer resulted in slower migration of the majorimmunoreactive peptides on polyacrylamide gels. Most of theimmunoreactive molecules migrated at 38-39 kD and less intense bandswere observed at 25 and 14 kD. It was necessary to run at least 10 timesas much reduced protein as nonreduced in order to detect the reducedmolecules. This is consistent with the affinity of the antibody formonomeric forms of the PDGF A and B chain peptides. These data indicatethat the major species in the PDGF-related affinity purified proteinsfrom conditioned media of HUVE cells was monomeric peptide whichmigrates on acrylamide gels at an apparent molecular weight of 36 kDnonreduced and 38 kD when reduced.

EXAMPLE 2 BIOLOGICAL ASSAYS

Chemotactic activity was determined in the Boyden chamber chemotaxisassay with NIH 3T3 or bovine aortic smooth muscle (BASM) cells asdescribed (Grotendorst, et al., Proc. Natl. Acad. Sci. USA,78:3669-3672, 1981; Grotendorst, et al., Methods in Enzymol.,147:144-152, 1987). Mitogenic assays were performed using 96 well platesand normal rat kidney (NRK) fibroblasts or NIH 3T3 cells as targetcells. The cells were plated in DMEM, 10% FCS; NRK cell cultures wereused 10-14 days after confluence and 3T3 cells made quiescent byincubation for 2 days in serum-free DMEM, 0.2 mg/ml BSA before use.Sample proteins and dilutions of known standards were added to the wellsand the plates incubated at 37° C. in 10% CO₂, 90% air for 18 hours,after which ³ H-thymidine at a final concentration of 5 uCi/ml was addedand incubated for an additional 2 hours. The media was removed, thecells washed and DNA synthesis determined from the ³ H-thymidineincorporation into trichloroacetic acid precipitable material byscintillation counting.

Gel Electrophoresis and Immuneblotting

Electrophoresis was performed on 12% polyacrylamide gels containing SDS(Laemmli, U.K., Nature, 227:680-685, 1970) unless otherwise stated.Immunoblotting was performed by electroblotting the proteins to anitrocellulose membrane and incubating the membrane in 50 mM Tris-HCI,pH 7.4, 100 mM NaCI (TBS) with 5% non-fat dry milk at 25° C. for 1 hourto block non-specific antibody binding. The blocking solution wasremoved and the antibody (15/μg/ml) added in TBS containing 0.5% non-fatdry milk and 1 μg/ml sodium azide and incubated overnight at 25° C. Themembranes were washed 5 times in TBS, 0.5% milk for 10 minutes each washand then incubated with alkaline phosphatase conjugated affinitypurified rabbit anti-goat IgG (KPL, Gaithersburg, Md.) at a 1:1000dilution in TBS containing 0.5% milk at 25° C. for 1 hour. The filterswere washed with TBS five times, 10 minutes each time, and the blotdeveloped using an alkaline phosphatase substrate solution (0.1 MTris-HCI, pH 9, 0.25 mg/ml nitro blue tetrazolium, 0.5 mg/ml 5bromo-4-chloro-3-indolyl phosphate).

Major Chemotactic and Mitogenic Activity is Produced by 36 kD Peptideand Not PDGF Peptides

In order to determine if the chemotactic and mitogenic activitiesobserved in the partially purified media proteins were from moleculescontaining the PDGF A and B chain peptides or were the products ofmolecules which do not contain these sequences, biological assays wereperformed with serial dilutions of the affinity purified media proteinsand serial dilutions of recombinant PDGF AA and BB homodimers and the ABheterodimer. Sufficient quantities of the samples were prepared toperform the mitogenic and chemotactic assays and the immunoblots withaliquots of each dilution sample. The mitogenic activity of the HUVEaffinity purified factors observed was comparable to the activityelicited by all three recombinant PDGF dimers. The chemotactic activitywas comparable to the AB heterodimer, producing less response than theBB homodimer and greater response than the AA homodimer. When thebiological activity of the samples was compared with immunoblots ofequivalent amounts of the same samples, no A chain nor B chain moleculeswere detected in the test samples. These data demonstrate the majorbiological activity present in the anti-PDGF affinity purified fractioncannot be accounted for by PDGF A or B chain containing molecules andimply that the major PDGF-immunoreactive protein species present inthese samples (the 36 kD peptide) is biologically active and does notcontain amino acid sequences found in the amino and carboxy terminals ofthe PDGF A or B chain peptides.

EXAMPLE 3 RECEPTOR COMPETITION ASSAYS

Assays were performed using confluent cultures of NIH 3T3 cells in 24well plates (Costar) grown in DMEM, 10% fetal calf serum, 10 μg/mlGentamicin. The growth media was removed and the cells washed twice withserum-free DMEM, 0.2 mg/ml BSA and the plates placed on ice for 30minutes in serum-free DMEM, 0.2 mg/ml BSA. Test samples and controlswere made up in serum-free DMEM, 0.2 mg/ml BSA containing 5-10 ng/ml ofHUVE affinity purified proteins and a serial dilution of one of therecombinant PDGF isoforms in a concentration range of 300 ng/ml to 16ng/ml. One milliliter aliquots of the samples were placed into wells ofthe 24 well plates and incubated on ice on a platform rocker for twohours. After the incubation period, the cells were washed three timesfor 10 minutes each on ice with PBS. The proteins bound to the surfaceof the cells were eluted with 5 ul of 1N acetic acid for 10 minutes. Theacetic acid elution samples were lyophilized, resuspended in 5 mM HCL,run on 12% polyacrylamide gels and immunoblotted to nitrocellulose usingthe anti-PDGF antibody.

In order to substantiate the binding of the endothelial cell moleculesto the PDGF cell surface receptors, competitive receptor binding assayswere performed. Because immunoblots of the affinity purified HUVE cellsecreted proteins indicated the presence of multiple PDGF immunoreactivemolecules, ¹²⁵ -I-labeled PDGF competition assays could not be usedsince this would not indicate which molecules in this mixture werecompeting for binding of the labeled PDGF for the receptors on thetarget cells. Since the isoforms of PDGF and the major PDGFimmunorelated protein secreted by HUVE cells are of different molecularweights, receptor binding competition was demonstrated on immunoblots.Direct binding of the anti-PDGF immunoreactive peptides to NIH 3T3 cellswas demonstrated by incubating monolayers of the 3T3 fibroblasts withthe anti-PDGF affinity purified proteins (10 ng/ml) for 2 hours at 4° C.Bound peptides were released by washing of the cell layer with 1N aceticacid and quantitated by immunoblot analysis using anti-PDGF IgG. Thisdata show that the 36 kD immunoreactive peptide binds to cell surface ofNIH 3T3 cells. This binding can be competed by increasing concentrationsof recombinant PDGF BB added to the binding media. These data suggestthat the CTGF peptide binds to specific cell surface receptors on NIH3T3 cells and that PDGF BB can compete with this binding.

RNA Isolation and Northern Blotting

Total RNA was isolated from cells in monolayer culture cells.Lyophilized RNA was resuspended in gel loading buffer containing 50%formamide and heated at 95° C. for two minutes before loading (20/μg perlane total RNA) onto 2.2M formaldehyde, 1% agarose gels and run at 50volts. Integrity of RNA was determined by ethidium bromide staining andvisualization of 18S and 28S rRNA bands. After electrophoresis the RNAwas transferred to nitrocellulose by blotting overnight with 10X SSCbuffer. The nitrocellulose was air dried and baked at 80° C. for 2 hoursin a vacuum oven. Hybridization was performed overnight at 46° C. withthe addition of 5×10⁵ CPM per ml of ³² P-labeled probe. Normally forNorthern blots, the entire plasmid was labeled and used as a probe.Labeling was done with a random primer labeling kit from BoehringerMannhelm. After hybridization, membranes were washed twice in 2X SSC,0.1% SDS for 15 minutes each at room temperature, once for 15 minutes in0.1X SSC, 0.1% SDS, room temperature and a final 15 minutes wash in 0.1XSSC, 0.1% SDS at 46° C. Blots were autoradiographed at -70° C. on KodakX-omar film.

EXAMPLE 4 LIBRARY SCREENING, CLONING, AND SEQUENCING

Standard molecular biology techniques were used to subclone and purifythe various DNA clones (Sambrook, et al., Molecular Cloning a LaboratoryManual, Second edition, Cold Spring Harbor Laboratory Press, Col. SpringHarbor, N.Y.). Clone DB60 was picked from a lambda gt11 HUVE cell cDNAlibrary by induction of the fusion proteins and screening with anti-PDGFantibody. Plaques picked were rescreened and positive clones replated atlow titer and isolated.

The EcoR I insert from clone DB60 was cloned into the M13 phage vectorand single-stranded DNA obtained for clones with the insert in oppositeorientations. These M13 clones were then sequenced by the dideoxy methodusing the Sequenase kit (U.S. Biochemical) and ³⁵ S-dATP (duPont). Bothstrands of DNA for this clone were completely sequenced using primerextension and both GTP and ITP chemistry. Aliquots of the sequencingreactions were run on both 6% acrylamide (16 hours) and 8% acrylamide (6hours) gels, vacuum dried and autoradiographed for at least 18 hours.

The cDNA fragment from clone DB60 was ³² P-CTP labeled and used torescreen the HUVE cell cDNA lambda gt11 library. Several clones werepicked and the largest, the 2100 bp clone designed DB60R32, wassubcloned into Bluescript phagemid. Subclones were made of Pst I, Kpn I,and Eco RI/Kpn I restriction fragments also in Bluescript. Thesesubclones were sequenced by double-stranded plasmid DNA sequencingtechniques using Sequenase as described above. The 1458 bp Eco RI/Kpn Iclone containing the open reading frame was subcloned into M13 mp18 andM13 mp19 and both strands of DNA were completely sequenced usingsingle-stranded DNA sequencing techniques with primer extension and bothGTP and ITP chemistry.

Cloning Expression and Sequencing of the cDNA for Connective TissueGrowth Factor

In order to further characterize these PDGF related molecules,sufficient quantities of the CTGF protein for amino acid sequencing wasneeded. However, the low concentrations of CTGF in the conditioned mediaof HUVE cell cultures and the costly and time consuming techniquesinvolved in obtaining and culturing these cells made proteinpurification to homogeneity and amino acid sequencing impractical.Therefore, the anti-PDGF antibody was used to screen an HUVE cell cDNAlibrary made in the expression vector lambda gt11. Over 500,000recombinant clones were screened. Several clones which gave strongsignals with the anti-PDGF antibody in the screening process werepurified and subcloned into the M13 phage vector and partial sequencedata obtained by single-stranded DNA sequencing. A search of the GenBankDNA sequence data base indicated that two of the clones picked containedfragments of the PDGF B chain cDNA open reading frame sequence. One ofthese clones was similar to a 1.8 kb insert previously isolated byCollins, et al. (Nature, 316:748-750, 1985) using a c-sis cDNA probe. Athird clone of 500 bp was completely sequenced and no match was found ina homology search of all nucleotide and amino acid sequences in GenBank(CEF 10 sequence was not available at that time). This clone wasdesignated DB60. Anti-PDGF antibody binding to the fusion proteinproduced by the clone DB60 was completely blocked by the affinitypurified proteins. A ³² P-labeled probe was made of DB60 and used on aNorthern blot of 20 μg of total RNA isolated from HUVE cells. The blotindicated probe hybridization with an mRNA of 2.4 kilobases, which is amessage of sufficient size to produce the proteins in the 38 kDmolecular weight range seen on the immunoblots of the affinity purifiedproteins. The DB60 clone was used to rescreen the HUVE cell cDNA lambdagt11 library and the largest clone isolated contained a 2100 base pairinsert designated DB60R32. A probe made with the 2100 bp Eco RI insertof clone DB60R32 also hybridized with a single 2.4 kb message in aNorthern blot of total RNA from HUVE cells.

EXAMPLE 5 IN VITRO TRANSCRIPTION AND TRANSLATION

In vitro transcription reactions were done using the 2100 bp cDNA cloneDB60R32 in the Bluescript KS vector. The plasmid was cut with Xho Iwhich cuts the plasmid once in the multiple cloning site of the vector3' to the cDNA insert. The T7 promoter site located 5' to the cDNAinsert was used for transcription. The in vitro transcriptions were donewith a kit supplied with the Bluescript vector (Stratagene).

In vitro translation reactions were done using nuclease treated rabbitreticulocyte lysate and ³⁵ S-cysteine in a cysteine-free amino acid mixfor labeling of the peptide (Promega). The reactions were done in afinal volume of 50 ul containing ³⁵ S-cysteine 1 mCi/ml (1200 Ci/mMole,DuPont), and serial dilutions of mRNA from the in vitro transcriptionreactions in concentrations ranging from 50 to 500 nanograms perreaction tube. The reactions were incubated at 30° C. for 60 minutes.Aliquots of the reactions were run on reduced or nonreduced 12%polyacrylamide electrophoresis gels, dried, and autoradiographed.

Bacterial expression of immunoreactive CTGF peptide was accomplished bysubcloning clone DB60R32 into the Eco RI site of the pET 5 expressionvector (Studier, et al., Ed. Academic Press, N.Y. Vol. 185, 60-89, 1990)in both sense and inverse orientations (as determined by restrictionenzyme digest analysis). Cultures of E. coli HMS174 cells were grown inM9 media to an OD 600 of 0.7 and the media made 0.4 mM IPTG andincubation continued for 2 hours. The cells were pelleted, lysed,inclusion bodies removed by centrifugation and aliquots of the pelletextracts run on 12% polyacrylamide gels and immunoblotted using theanti-PDGF antibody. The protein produced by clone DB60R32 in the senseorientation produced anti-PDGF immunoreactive peptides in the 36-39 kDMW range while the anti-sense control produced no immunoreactivepeptides. The recombinant peptides produced in the E. Coli systemcompletely blocked the anti-PDGF reaction with the CTGF peptides presentin conditioned media.

Expression of CTGF in Xenopus

For expression in Xenopus oocytes, mature X. laevis females wereobtained from Nasco (Fort Atkinson, Wis.) and maintained at roomtemperature. Frogs were anesthetized by hypothermia and the ovariantissue was surgically removed. Ovarian tissue was minced and digestedthe 0.2% collagenase (Sigma Type II)in OR-2 without calcium (Wallace, etal., Exp. Zool., 184:321-334, 1973) for 2-3 hours. Unblemished stage VIoocytes (Dumont, J. Morphol., 136:153-180, 1972), 1.3 mm diameter, werethen carefully selected and microinjected.

Stage VI oocytes (5-10 at a time) were placed on a hollowed plexiglassplatform and drained of excess OR-2 solution. Approximately 50 nl ofsample containing 10 ng of RNA was injected into the animal pole justabove the oocyte equator using a Leitz system microinjector. Followinginjection, oocytes were returned to OR-2 buffer with 0.1% BSA andincubated for 24 hours at 25° C. Viable oocytes were then pooled andextracted by homogenization in 100 mm NaCI, 10 mm Tris pH 7.5 with tenstrokes of a Dounce homogenizer (20 μl/oocyte). The homogenate was thenmixed with an equal volume of freon to remove pigment and lipid andcentrifuged at 10,000 rpm for 30 seconds to separate the phases. The topaqueous phase was removed and tested for chemotactic activity using NIH3T3 cells as described above.

Injection of Xenopus oocytes with 10 ng of RNA preparations derived byin in vitro transcription of the DB60 R32 clone resulted in theproduction of a fibroblast chemotactic activity. Control injected cellsdid not produce this activity. These results indicate that the openreading frame of the DB60 R32 clone encodes a protein with chemotacticactivity for fibroblastic cells as does CTGF.

EXAMPLE 6 SEQUENCE ANALYSIS OF CTGF

The 2100 bp insert of clone DB60R32 was sequenced initially bysubcloning of Pst I and Kpn I restriction fragments into Bluescript andusing double-stranded dideoxy methods. This indicated an open readingframe of 1047 base pairs and oriented the DB60 insert to the largercDNA. An Eco RI/Kpn I fragment containing the entire open reading framewas inserted into M13 mp18 and M13 mp19 and both strands of the DNA weresequenced with single-stranded dideoxy methods by primer extension usingboth GTP and the GTP analog ITP. The cDNA nucleotide sequence of theopen reading frame encoded a 38,000 MW protein, confirming the cell-freetranslation results and matching the size of the immunopurifiedpeptides. A new search of the GenBank data base revealed that this cDNAhad a 50% nucleotide sequence homology with CEF-10 mRNA, one of theimmediate early genes induced in v-src transformed chicken embryofibroblasts (Simmons, et al., Proc. Natl. Acad. Sci. USA, 86:1178-1182,1989). The translated cDNA for human CTGF and avian CEF-10 have a 45%overall homology and a 52% homology if the putative alternative splicingregion is deleted. This region is between amino acids 171 (asparticacid) and 199 (cysteine) in the CTGF sequence.

Although the invention has been described with reference to thepresently preferred embodiment, it should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the invention is limited only by the followingclaims.

SUMMARY OF SEQUENCES

Sequence ID No. 1 is the nucleic acid sequence (and the deduced aminoacid sequence) of cDNA encoding CTGF of the present invention.

Sequence ID No. 2 is the deduced amino acid sequence of CTGF of thepresent invention.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 2                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2075 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (vii) IMMEDIATE SOURCE:                                                       (B ) CLONE: DB60R32                                                           (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 130..1177                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       CCCGGCCGACAGCCCCGAGACGACAGCCCGGCGCGTCCCGGTCCCCACCTCCGACCACCG60                CCAGCGCTCCAGGCCCCGCGCTCCCCGCTCGCCGCCACCGCGCCCTCCGCTC CGCCCGCA120              GTGCCAACCATGACCGCCGCCAGTATGGGCCCCGTCCGCGTCGCCTTC168                           MetThrAlaAlaSerMetGlyProValArgValAlaPhe                                       15 10                                                                         GTGGTCCTCCTCGCCCTCTGCAGCCGGCCGGCCGTCGGCCAGAACTGC216                           ValValLeuLeuAlaLeuCysSerArgProAlaValGlyGlnAsnCys                              152025                                                                        AGC GGGCCGTGCCGGTGCCCGGACGAGCCGGCGCCGCGCTGCCCGGCG264                          SerGlyProCysArgCysProAspGluProAlaProArgCysProAla                              30354045                                                                       GGCGTGAGCCTCGTGCTGGACGGCTGCGGCTGCTGCCGCGTCTGCGCC312                          GlyValSerLeuValLeuAspGlyCysGlyCysCysArgValCysAla                              505560                                                                        AAGCAGCTGGGCGAGCTGTGCACCGAGCGCGACCCCTGCGACCCGCAC360                           LysGlnLeuGlyGluLeuCysThrGluArgAspProCysAspProHis                              657075                                                                        A AGGGCCTCTTCTGTGACTTCGGCTCCCCGGCCAACCGCAAGATCGGC408                          LysGlyLeuPheCysAspPheGlySerProAlaAsnArgLysIleGly                              808590                                                                        GTGTGC ACCGCCAAAGATGGTGCTCCCTGCATCTTCGGTGGTACGGTG456                          ValCysThrAlaLysAspGlyAlaProCysIlePheGlyGlyThrVal                              95100105                                                                      TACCGCAGCGGAGAG TCCTTCCAGAGCAGCTGCAAGTACCAGTGCACG504                          TyrArgSerGlyGluSerPheGlnSerSerCysLysTyrGlnCysThr                              110115120125                                                                  TGCCTGGACGG GGCGGTGGGCTGCATGCCCCTGTGCAGCATGGACGTT552                          CysLeuAspGlyAlaValGlyCysMetProLeuCysSerMetAspVal                              130135140                                                                     CGTCTGCCCA GCCCTGACTGCCCCTTCCCGAGGAGGGTCAAGCTGCCC600                          ArgLeuProSerProAspCysProPheProArgArgValLysLeuPro                              145150155                                                                     GGGAAATGCTGC GAGGAGTGGGTGTGTGACGAGCCCAAGGACCAAACC648                          GlyLysCysCysGluGluTrpValCysAspGluProLysAspGlnThr                              160165170                                                                     GTGGTTGGGCCTGCCCTC GCGGCTTACCGACTGGAAGACACGTTTGGC696                          ValValGlyProAlaLeuAlaAlaTyrArgLeuGluAspThrPheGly                              175180185                                                                     CCAGACCCAACTATGATTAGAGCCAA CTGCCTGGTCCAGACCACAGAG744                          ProAspProThrMetIleArgAlaAsnCysLeuValGlnThrThrGlu                              190195200205                                                                  TGGAGCGCCTGTTCCAAGACCT GTGGGATGGGCATCTCCACCCGGGTT792                          TrpSerAlaCysSerLysThrCysGlyMetGlyIleSerThrArgVal                              210215220                                                                     ACCAATGACAACGCCTCCTGC AGGCTAGAGAAGCAGAGCCGCCTGTGC840                          ThrAsnAspAsnAlaSerCysArgLeuGluLysGlnSerArgLeuCys                              225230235                                                                     ATGGTCAGGCCTTGCGAAGCTGAC CTGGAAGAGAACATTAAGAAGGGC888                          MetValArgProCysGluAlaAspLeuGluGluAsnIleLysLysGly                              240245250                                                                     AAAAAGTGCATCCGTACTCCCAAAATCTC CAAGCCTATCAAGTTTGAG936                          LysLysCysIleArgThrProLysIleSerLysProIleLysPheGlu                              255260265                                                                     CTTTCTGGCTGCACCAGCATGAAGACATACCGAGCTA AATTCTGTGGA984                          LeuSerGlyCysThrSerMetLysThrTyrArgAlaLysPheCysGly                              270275280285                                                                  GTATGTACCGACGGCCGATGCTGCACCCCCCAC AGAACCACCACCCTG1032                         ValCysThrAspGlyArgCysCysThrProHisArgThrThrThrLeu                              290295300                                                                     CCGGTGGAGTTCAAGTGCCCTGACGGCGAGGTC ATGAAGAAGAACATG1080                         ProValGluPheLysCysProAspGlyGluValMetLysLysAsnMet                              305310315                                                                     ATGTTCATCAAGACCTGTGCCTGCCATTACAACTG TCCCGGAGACAAT1128                         MetPheIleLysThrCysAlaCysHisTyrAsnCysProGlyAspAsn                              320325330                                                                     GACATCTTTGAATCGCTGTACTACAGGAAGATGTACGGAG ACATGGCAT1177                        AspIlePheGluSerLeuTyrTyrArgLysMetTyrGlyAspMetAla                              335340345                                                                     GAAGCCAGAGAGTGAGAGACATTAACTCATTAGACTGGAACTTGAACTGATTCACATCT C1237             ATTTTTCCGTAAAAATGATTTCAGTAGCACAAGTTATTTAAATCTGTTTTTCTAACTGGG1297              GGAAAAGATTCCCACCCAATTCAAAACATTGTGCCATGTCAAACAAATAGTCTATCTTCC1357              CCAGACACTGGTTTGAAGAATGTTAAGACTTGACAG TGGAACTACATTAGTACACAGCAC1417             CAGAATGTATATTAAGGTGTGGCTTTAGGAGCAGTGGGAGGGTACCGGCCCGGTTAGTAT1477              CATCAGATCGACTCTTATACGAGTAATATGCCTGCTATTTGAAGTGTAATTGAGAAGGAA1537              AATTTTAGCGTGC TCACTGACCTGCCTGTAGCCCCAGTGACAGCTAGGATGTGCATTCTC1597             CAGCCATCAAGAGACTGAGTCAAGTTGTTCCTTAAGTCAGAACAGCAGACTCAGCTCTGA1657              CATTCTGATTCGAATGACACTGTTCAGGAATCGGAATCCTGTCGATTAGACTGGACAGC T1717             TGTGGCAAGTGAATTTGCCTGTAACAAGCCAGATTTTTTAAAATTTATATTGTAAATATT1777              GTGTGTGTGTGTGTGTGTGTATATATATATATATATGTACAGTTATCTAAGTTAATTTAA1837              AGTTGTTTGTGCCTTTTTATTTTTGTTTTTAATGCT TTGATATTTCAATGTTAGCCTCAA1897             TTTCTGAACACCATAGGTAGAATGTAAAGCTTGTCTGATCGTTCAAAGCATGAAATGGAT1957              ACTTATATGGAAATTCTGCTCAGATAGAATGACAGTCCGTCAAAACAGATTGTTTGCAAA2017              GGGGAGGCATCAG TGTCTTGGCAGGCTGATTTCTAGGTAGGAAATGTGGTAGCTCACG2075               (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 349 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetThrAlaAlaSerMetGlyPr oValArgValAlaPheValValLeu                             151015                                                                        LeuAlaLeuCysSerArgProAlaValGlyGlnAsnCysSerGlyPro                              2025 30                                                                       CysArgCysProAspGluProAlaProArgCysProAlaGlyValSer                              354045                                                                        LeuValLeuAspGlyCysGlyCysCysArgValCysAlaLysGlnL eu                             505560                                                                        GlyGluLeuCysThrGluArgAspProCysAspProHisLysGlyLeu                              65707580                                                                      PheCysAspPhe GlySerProAlaAsnArgLysIleGlyValCysThr                             859095                                                                        AlaLysAspGlyAlaProCysIlePheGlyGlyThrValTyrArgSer                              100 105110                                                                    GlyGluSerPheGlnSerSerCysLysTyrGlnCysThrCysLeuAsp                              115120125                                                                     GlyAlaValGlyCysMetProLeuCysSerMetAs pValArgLeuPro                             130135140                                                                     SerProAspCysProPheProArgArgValLysLeuProGlyLysCys                              145150155160                                                                   CysGluGluTrpValCysAspGluProLysAspGlnThrValValGly                             165170175                                                                     ProAlaLeuAlaAlaTyrArgLeuGluAspThrPheGlyProAspPro                               180185190                                                                    ThrMetIleArgAlaAsnCysLeuValGlnThrThrGluTrpSerAla                              195200205                                                                     CysSerLysThrCysGlyMetGly IleSerThrArgValThrAsnAsp                             210215220                                                                     AsnAlaSerCysArgLeuGluLysGlnSerArgLeuCysMetValArg                              225230235 240                                                                 ProCysGluAlaAspLeuGluGluAsnIleLysLysGlyLysLysCys                              245250255                                                                     IleArgThrProLysIleSerLysProIleLysPheGluLeuSe rGly                             260265270                                                                     CysThrSerMetLysThrTyrArgAlaLysPheCysGlyValCysThr                              275280285                                                                     AspGlyArgCys CysThrProHisArgThrThrThrLeuProValGlu                             290295300                                                                     PheLysCysProAspGlyGluValMetLysLysAsnMetMetPheIle                              305310 315320                                                                 LysThrCysAlaCysHisTyrAsnCysProGlyAspAsnAspIlePhe                              325330335                                                                     GluSerLeuTyrTyrArgLysMetTyrGlyAsp MetAla                                      340345                                                                    

We claim:
 1. Substantially pure connective tissue growth factor (CTGF)polypeptide, wherein the polypeptide is characterized by:(a) mitogenicand chemotactic activity for connective tissue cells; (b) a molecularweight of approximately 36 kD by non-reducing SDS-PAGE and approximately38 kD by reducing SDS-PAGE; (c) binding to a PDGF receptor; (d) existingas a monomer; and (e) a polypeptide sequence according to Sequence IDNo.
 2. 2. Antibodies which bind the CTGF polypeptide of Sequence IDNo.2, but not platelet derived growth factor (PDGF).
 3. The antibodiesof claim 2, wherein the antibodies are polyclonal.
 4. The antibodies ofclaim 2, wherein the antibodies are monoclonal.